Preparation method and application of natural active ingredient compound nanogel for veterinary use

Abstract

The application discloses a preparation method and application of a natural active ingredient compound nanogel agent for animals, and belongs to the technical field of veterinary drug preparations. The natural active ingredient compound nanogel agent disclosed by the application is composed of natural active ingredients, a gel matrix and additional agents, is reasonable in formula, simple in process, stable in property, convenient to use and safe and long-acting. The natural active ingredient compound nanogel agent contains two synergistically acting natural active ingredients, can inhibit the proliferation of endometritis pathogenic bacteria such as staphylococcus aureus and streptococcus, and reduce the expression of pro-inflammatory cytokines at an inflammation site; meanwhile, the natural active ingredient compound nanogel agent has a certain slow-release capacity, slowly releases natural active ingredient nanoparticles easy to be taken by cells, can realize long-time residence and high-efficiency absorption of drugs at a drug administration site, and thus can enhance the action time, reduce the drug administration frequency and improve the curative effect.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug formulation technology, and particularly relates to a method for preparing and applying a compound nanogel of natural active ingredients for veterinary use. Background Technology

[0002] Reproduction is a fundamental characteristic of animals and is crucial for the survival of the species. Uterine bacterial infections can lead to numerous problems in animals, including decreased reproductive performance, infertility, and genetic loss, severely reducing reproductive capacity and increasing breeding costs, resulting in significant economic losses for the livestock industry. Therefore, achieving efficient prevention and control of uterine bacterial infections has become an important means to improve the economic efficiency of the livestock industry.

[0003] Studies have shown that pathogenic microorganisms can induce abnormal expression of various chemokines and adhesion factors in endometrial cells, thereby reducing endometrial receptivity and leading to infertility and miscarriage. Simultaneously, the immune system is activated by pathogens, and the production of various inflammatory factors can inhibit the synthesis and secretion of gonadotropins. The large amounts of reactive oxygen species (ROS) produced by mitochondria in phagocytes during the inflammatory response can directly or indirectly activate multiple inflammatory signaling pathways, exacerbating the inflammatory response and affecting processes such as oocyte maturation, embryo implantation, fetal development, and uterine contractions. Furthermore, persistent inflammation can lead to mucosal ulcers and erosions, and even intrauterine adhesions or uterine polyps, severely impairing the body's reproductive function. Therefore, effective antibacterial and anti-inflammatory treatments are crucial strategies for treating bacterial infections of the uterus. Currently, the combined use of anti-inflammatory hormones and antibiotics has become the mainstream treatment for bacterial infections of the uterus. However, this approach can induce bacterial resistance, resulting in antibiotic residues and affecting the quality of animal products. At the same time, traditional hormonal drugs are prone to causing metabolic disorders, complications, immunosuppression, and food safety issues. Therefore, finding novel, safe, and highly effective alternative drugs with low bacterial resistance is particularly important and urgent.

[0004] To advance the prevention and treatment of bacterial infections of the uterus, researchers aim to develop convenient, safe, efficient, and long-lasting natural active ingredient nanogels by combining natural active ingredients with synergistic antibacterial and anti-inflammatory capabilities, based on self-assembled nanotechnology and gel formulation. However, most natural active ingredients have extremely poor water solubility, which significantly limits their absorption at the administration site, resulting in less than ideal bioavailability. Self-assembled nanoparticles represent a potential delivery system for promoting transmembrane drug transport.

[0005] Among common dosage forms for treating bacterial infections of the uterus, oral formulations have poor bioavailability, injections have poor targeting and are inconvenient to administer, suppositories have low drug loading, and irrigates are prone to leakage into the vagina. Compared with the above-mentioned formulations, gels have better tissue adhesion and sustained-release properties, enabling high drug concentrations and prolonged residence at the administration site. For example, the catechol groups or amino groups in chitosan gel structures can interact with tissues, allowing the gel to adhere to the tissue surface. Zhi Weiwei et al. placed a thermosensitive gel loaded with berberine hydrochloride at 37°C, and its release rate was about 60% after 12 hours, while the release rate of free berberine hydrochloride was close to 100% after 2 hours. In addition, good biocompatibility, biodegradability, and suitability for vaginal administration also ensure the safe application of gels. Therefore, as an efficient delivery strategy, gels have good prospects for uterine drug delivery.

[0006] Although research on natural active ingredients is developing rapidly in various fields, related research in the treatment of bacterial infections of the uterus has not been carried out on a large scale, which indicates that gels containing natural active ingredients suitable for uterine administration need further research and development and use. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention proposes a method for preparing and applying a compound nanogel containing natural active ingredients for veterinary use.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A veterinary natural active ingredient compound nanogel, wherein the veterinary natural active ingredient compound nanogel is composed of natural active ingredients, a gel matrix and additives; wherein the natural active ingredients are prepared by ultrasonic treatment of a first natural active ingredient and a second natural active ingredient.

[0010] As a further preferred embodiment, the concentration of the natural active ingredient is 3-4 wt%, the concentration of the gel matrix is ​​0.6-7.0 wt%, and the concentration of the additive is 0.03-15 wt%.

[0011] As a further preferred embodiment, the mass ratio of the first natural active ingredient and the second natural active ingredient is (1-2):1.

[0012] As a further preferred embodiment, the first natural active ingredient is rhein, glyceryl laurate, or pterostilbene, more preferably glyceryl laurate; the second natural active ingredient is magnolol or honokiol, more preferably honokiol.

[0013] As a further preferred embodiment, the gel matrix is ​​at least one of carbomer 940, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose and chitosan, more preferably carbomer 940.

[0014] As a further preferred embodiment, the additive is at least one of propylene glycol and ethylparaben, and the additive also includes triethanolamine for adjusting the pH.

[0015] This invention also provides a method for preparing the aforementioned veterinary natural active ingredient compound nanogel, comprising the following steps:

[0016] Natural active ingredients are added to anhydrous ethanol and hydrothermally prepared into a natural active ingredient nano suspension. Then, after the gel matrix is ​​fully swollen, it is added to the nano suspension along with the additives. The mixture is stirred and the pH is adjusted to 6.0-7.5 to obtain a veterinary natural active ingredient compound nano gel.

[0017] The present invention also provides the application of the aforementioned veterinary natural active ingredient compound nanogel in the preparation of a drug for treating uterine bacterial infections.

[0018] The present invention also provides the application of the aforementioned veterinary natural active ingredient compound nanogel in the preparation of a drug for relieving bacterial inflammatory uterine infections.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] This invention prepares a veterinary natural active ingredient compound nanogel, which innovatively combines natural active ingredients with synergistic antibacterial and anti-inflammatory effects to form small-diameter nanoparticles that are easily absorbed by the body. Specifically, glyceryl laurate can disrupt bacterial cell membrane and cell wall structures, inhibiting the activation of MAPK and NF-κB signaling pathways; while magnolol can cause reactive oxygen species to accumulate in bacterial mitochondria to kill bacteria, while simultaneously inhibiting inflammatory signaling pathways such as Klf4, p38, and ERK1 / 2. The combined effect of these two components leads to an imbalance in bacterial intracellular homeostasis and reduces the expression of pro-inflammatory cytokines, ultimately achieving highly effective antibacterial and anti-inflammatory efficacy, demonstrating good therapeutic ability against bacterial infections of the uterus. On the one hand, it avoids the problems of frequent drug resistance, antibiotic residues, metabolic disorders, complications, immunosuppression, and food safety issues caused by current antibiotic-hormone therapy; on the other hand, by forming small-diameter nanoparticles, it improves the ability of natural active ingredients to adhere and transport across the endometrium, and the sustained-release properties of the gel prolong the residence time of natural active ingredients in the uterus, ultimately improving the utilization rate of natural active ingredients. Meanwhile, this gel also demonstrated good efficacy in clearing bacteria from the uterus and suppressing inflammation, effectively alleviating tissue damage caused by infectious inflammation. Furthermore, the gel's preparation process is simple, the excipients are inexpensive and readily available, its properties are stable, and it is easy to use, providing a safe and effective new option for the treatment of uterine bacterial infections, and showing promising development prospects. Attached Figure Description

[0021] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0022] Figure 1 The results of the synergistic anti-inflammatory activity assay of the natural active ingredients in Example 1 are shown.

[0023] Figure 2 The images shown are scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the natural active ingredients in Example 2, where A is a scanning electron microscope image and B is a transmission electron microscope image.

[0024] Figure 3 This is a photograph of the veterinary natural active ingredient compound nanogel for Experimental Example 2.

[0025] Figure 4 The in vitro release curves of the veterinary natural active ingredient compound nanogel and the natural active ingredients are shown in Experiment Example 3. Among them, A is the in vitro release curve of glyceryl laurate, and B is the in vitro release curve of magnolol.

[0026] Figure 5 The anatomical morphology of the uterus of rats in each group on the 4th and 8th days of treatment;

[0027] Figure 6The bacterial load in the uterine tissue of rats in each group after treatment is shown in Figure 4, where A represents day 4 of treatment and B represents day 8 of treatment.

[0028] Figure 7 The values ​​represent the expression levels of inflammatory factors in the uterine tissue of rats in each group after treatment. A, B, C, and D represent day 4 of treatment, while E, F, G, and H represent day 8 of treatment.

[0029] Figure 8 The results of histopathological observation of uterine tissue in rats in each group on day 8 of treatment are shown. Among them, A is the blank control group, B is the infection control group, C is the raw material and drug combination group, and D is the veterinary natural active ingredient compound nanogel group. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0034] The terms "comprising," "including," "having," and "containing," etc., used in this document are open-ended, meaning they include but are not limited to. All raw materials used in the following embodiments of this invention are commercially available. Specifically, magnolol, hydroxypropyl methylcellulose, carbomer 940, and triethanolamine were purchased from Maclean's Reagent Company; magnolol, rhein, glyceryl laurate, and pterostilbene were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; anhydrous ethanol, propylene glycol, sodium carboxymethyl cellulose, and chitosan were purchased from Sinopharm Chemical Reagent Co., Ltd.; and ethylparaben was purchased from Aladdin Reagent Co., Ltd.

[0035] The following embodiments are further illustrations of the technical solution of the present invention.

[0036] To promote the prevention and treatment of uterine bacterial infections, this invention combines natural active ingredients with synergistic antibacterial and anti-inflammatory capabilities, and develops a convenient, safe, efficient, and long-lasting natural active ingredient nanogel based on self-assembled nanotechnology and gel formulation. The natural active ingredients are unlikely to cause complications, are non-irritating, do not exhibit cross-resistance, and leave virtually no residue. They kill bacteria by disrupting bacterial cell membranes and cell wall structures, inhibiting the expression of drug-resistant genes, increasing intracellular reactive oxygen species concentrations, and inhibiting bacterial biofilm formation. Simultaneously, they prevent the activation of NLRP3, NLRC4, and AIM2 inflammasomes to avoid pyroptosis, inhibit the activation of multiple signaling pathways such as MAPK, NF-κB, Klf4, p38, ERK1 / 2, JNK, and PI3K / Akt, and prevent the excessive release of pro-inflammatory cytokines such as IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-1β, and TNF-α. Using natural active ingredients to treat bacterial infections of the uterus instead of antibiotics and hormones can effectively exert synergistic antibacterial and anti-inflammatory effects while slowing down the development of bacterial resistance and reducing the risk of metabolic disorders and complications. For most natural active ingredients, the extremely poor water solubility significantly limits their transmembrane transport and absorption at the administration site. This invention improves their effective utilization rate by "modifying" them into self-assembled nanoparticles. Studies have shown that at the nanoscale, the smaller the particle size, the higher the dissolution rate and the easier it is for cells to take up. For example, the in vitro cellular absorption rate of lipid nanoparticles around 100 nm is lower than that of lipid nanoparticles around 400 nm. Clarithromycin nanocrystals, by reducing particle size, effectively promote their penetration and uptake within monolayer Caco-2 cells.

[0037] The specific technical solution is as follows:

[0038] This invention provides a veterinary natural active ingredient compound nanogel, which is composed of natural active ingredients, a gel matrix, and additives.

[0039] The natural active ingredient is prepared by ultrasonic treatment of the first natural active ingredient and the second natural active ingredient. The specific preparation method is as follows: (1) Add the first natural active ingredient and the second natural active ingredient to anhydrous ethanol, and sonicate for 10-15 minutes to promote dissolution, thereby obtaining the natural active ingredient.

[0040] In some preferred embodiments of the present invention, the concentration of the natural active ingredient is 3-4 wt%, the concentration of the gel matrix is ​​0.6-7.0 wt%, and the concentration of the additive is 0.03-15 wt%.

[0041] In some preferred embodiments of the present invention, the mass ratio of the first natural active ingredient and the second natural active ingredient is (1-2):1. The first natural active ingredient is rhein, glyceryl laurate, or pterostilbene, more preferably glyceryl laurate; the second natural active ingredient is magnolol or honokiol, more preferably honokiol.

[0042] In some preferred embodiments of the present invention, the gel matrix is ​​at least one selected from carbomer 940, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, and chitosan, more preferably carbomer 940, with a mass concentration of 0.8 wt%. The additive is at least one selected from propylene glycol and ethylparaben, and the additive also includes triethanolamine for pH adjustment. Propylene glycol is used as a humectant, and ethylparaben is used as a preservative; both can be mixed separately with the gel matrix and the natural active ingredient, or simultaneously with both. The amount of propylene glycol added is 15% of the veterinary natural active ingredient compound nanogel; the amount of ethylparaben added is 0.03% of the veterinary natural active ingredient compound nanogel.

[0043] This invention also provides a method for preparing the aforementioned veterinary natural active ingredient compound nanogel, comprising the following steps:

[0044] Natural active ingredients are added to anhydrous ethanol and hydrothermally prepared into a natural active ingredient nano suspension. Then, after the gel matrix is ​​fully swollen, it is added to the nano suspension along with the additives. The mixture is stirred and the pH is adjusted to 6.0-7.5 to obtain a veterinary natural active ingredient compound nano gel.

[0045] The specific preparation method is as follows:

[0046] 1) The first and second natural active ingredients are mixed in anhydrous ethanol and reacted under hydrothermal conditions of 80℃ and 2400r / min for 80-100min to obtain a nano suspension.

[0047] 2) Add ethylparaben to propylene glycol and sonicate to dissolve, obtaining solution A;

[0048] 3) Dissolve the gel matrix in water, and after it swells completely, add propylene glycol. Then add it together with solution A into the nano suspension and stir at 800-1200 r / min for 3-5 h to obtain gel A.

[0049] 4) Add triethanolamine to gel A until the pH is adjusted to between 6.0 and 7.5 to obtain a compound nano gel containing natural active ingredients for veterinary use.

[0050] The present invention also provides the application of the aforementioned veterinary natural active ingredient compound nanogel in the preparation of a drug for treating uterine bacterial infections.

[0051] The present invention also provides the application of the aforementioned veterinary natural active ingredient compound nanogel in the preparation of a drug for relieving bacterial inflammatory uterine infections.

[0052] This invention utilizes natural active ingredients with combined antibacterial and anti-inflammatory functions to prepare a veterinary natural active ingredient compound nanogel with good therapeutic effects on bacterial infections of the uterus. The invention employs gel formulation technology to construct a highly efficient nano-drug sustained-release system, promoting the development of natural active ingredient gel formulations and advancing the clinical application of natural active ingredients.

[0053] The following embodiments provide a detailed description of the technical solution of the present invention.

[0054] Example 1: In vitro combined antibacterial and synergistic anti-inflammatory effects of natural active ingredients

[0055] 1.1 In vitro combined antibacterial test of natural active ingredients

[0056] The various natural active ingredients are shown in Table 1. The relationship between the two natural active ingredients was determined by the fractional inhibitory concentration (FIC). FIC≤0.5, 0.5<FIC≤1, 1<FIC≤2, and FIC>2 represent synergistic, additive, unrelated, and antagonistic effects, respectively.

[0057] Table 1 Natural Active Ingredients

[0058]

[0059] The specific steps are as follows:

[0060] (1) Each natural active ingredient was serially diluted with LB broth medium to obtain 7 dilution concentrations of 1024 μg / mL, 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL and 16 μg / mL;

[0061] (2) Take 50 μL of each of the two corresponding natural active ingredients and arrange them in rows and columns of a 96-well plate. Add 100 μL of diluted bacterial solution to each well to make the final bacterial inoculum 5 × 10⁻⁶. 5 CFU / mL, incubate overnight.

[0062] (3) Calculate the FIC of the natural active ingredient. FIC = A (Minimum effective concentration of the first natural active ingredient when used in combination) / B (MIC of the first natural active ingredient when used alone) + C (Minimum effective concentration of the second natural active ingredient when used in combination) / D (MIC of the second natural active ingredient when used alone). (MIC in medicine refers to the lowest drug concentration that inhibits visible bacterial growth).

[0063] 1.2 FIC test results of various natural active ingredients

[0064] Table 2. FIC of each natural active ingredient against Staphylococcus aureus in Example 1

[0065]

[0066] Table 3. FIC of each natural active ingredient against Streptococcus in Example 1

[0067]

[0068] As can be seen from Tables 2 and 3, the FIC of glyceryl laurate combined with magnolol and glyceryl laurate combined with magnolol is the lowest against Staphylococcus aureus and Streptococcus, which means that the combined inhibitory effect of glyceryl laurate and magnolol is the strongest.

[0069] 1.3 Synergistic Anti-inflammatory Test of Natural Active Ingredients

[0070] Using glyceryl laurate and magnolol, and glyceryl laurate and magnolol as natural active ingredients, their in vitro anti-inflammatory capabilities were investigated through in vitro anti-inflammatory tests.

[0071] The specific steps are as follows:

[0072] (1) RAW264.7 cells were incubated for 24 h, the supernatant was discarded, and DMEM high glucose medium containing 10% fetal bovine serum was added. The cells were purged and mixed evenly, and seeded into 6-well plates at 2 mL / well. The cells were cultured for 24 h.

[0073] (2) Set up a normal group, an LPS (lipopolysaccharide) group, a single natural active ingredient group, and a combination group. Among them, the normal group was cultured with 2 mL of PBS for 24 h; the LPS group was cultured with 2 mL of LPS solution (0.1 μg / mL) for 2 h, and then replaced with 2 mL of PBS for 22 h; the single natural active ingredient group and the combination group were pre-added with 2 mL of LPS solution (0.1 μg / mL), incubated for 2 h, and then replaced with 2 mL of PBS containing different concentrations of natural active ingredients for 22 h.

[0074] (3) Divide the cells, centrifuge and collect the supernatant. Use the corresponding ELISA kit according to the instructions to detect the concentrations of TNF-α, IL-1β, IL-6 and IL-8 in the supernatant.

[0075] 1.4 Results of the test on the synergistic anti-inflammatory ability of natural active ingredients

[0076] Figure 1 This is the result of the synergistic anti-inflammatory ability test of natural active ingredients. It can be seen that, compared with the lipopolysaccharide group, the expression levels of the four pro-inflammatory cytokines decreased in all experimental groups. Meanwhile, glyceryl laurate, magnolol, and honokiol each have certain anti-inflammatory effects on their own, and their anti-inflammatory ability is further enhanced when combined. Compared with the two combined groups, the combination of glyceryl laurate and honokiol showed a stronger inhibitory effect on the expression levels of the four pro-inflammatory cytokines, indicating a better synergistic anti-inflammatory ability.

[0077] Based on the combined antibacterial and synergistic anti-inflammatory results, the natural active ingredients were ultimately selected as glyceryl laurate and magnolol in combination.

[0078] Example 2: Preparation of Natural Active Ingredient Nanoparticle Suspension and PDI and Particle Size Detection

[0079] The preparation conditions of the natural active ingredient nano-suspension are shown in Table 4.

[0080] Table 4. Preparation conditions of nano-suspensions of natural active ingredients

[0081]

[0082] The specific preparation steps are as follows:

[0083] (1) Add 330.0 mg of magnolol and 660.0 mg of glyceryl laurate to 10 mL of anhydrous ethanol, and sonicate for 10 min to promote dissolution, to obtain solution A.

[0084] (2) Stir solution A under the conditions shown in Table 4 for 90 min, adding 15 mL of distilled water during the process. In the last 30 min, open the stopper to evaporate the ethanol and obtain the natural active ingredient nano suspension prepared under different conditions.

[0085] The prepared natural active ingredient nano-suspensions were numbered 1-6 sequentially. 1 mL of each sample was taken, diluted 10-fold with distilled water, and stored in 10 mL centrifuge tubes for later use. The polydispersity index (PDI) and particle size were determined using a Malvern nanolaser particle size analyzer ZS90 at 25°C.

[0086] 2.2 Results of PDI and particle size analysis of natural active ingredient nano-suspension

[0087] The results of particle size analysis of the natural active ingredient nanosuspension PDI prepared in Example 2 are shown in Table 5.

[0088] Table 5. PDI and particle size of the natural active ingredient nanosuspension in Example 2

[0089]

[0090]

[0091] As shown in the test results of groups 1, 2, and 3 in Table 5, when the rotation speed is fixed at 2400 r / min, the higher the temperature, the smaller the nanoparticle size. However, as shown in the test results of groups 4, 5, and 6 in Table 5, when the temperature is fixed at 80℃, the PDI of the nanoparticles decreases with increasing rotation speed. This result indicates that a higher rotation speed results in more uniform nanoparticle size. Furthermore, although the PDI of the nanoparticles in the 1600 r / min group is lower than that in the 2400 r / min group, the difference is not significant, and the nanoparticle size in the 1600 r / min group is larger than that in the 2400 r / min group (groups 2 and 6 are two batches of experiments; the difference in data is due to data fluctuations caused by objective factors during the experiment, which is within a reasonable range. Subsequent experiments also had the same issue, which will not be elaborated further).

[0092] Therefore, 80℃ and 2400r / min were chosen as the optimal conditions for the preparation of the natural active ingredient nano-suspension.

[0093] Example 3: Screening and Viscosity Measurement of Gel Matrix

[0094] 3.1 Preparation of different blank gelling agents

[0095] The types and concentrations of pre-screened gel matrices are shown in Table 6, and blank gels of different types and concentrations are prepared accordingly.

[0096] Table 6. Types and concentrations of pre-screened gel matrices

[0097]

[0098] The specific preparation steps are as follows: the relevant tests for each concentration are shown in (1)-(12).

[0099] A. (1) Weigh 125.0 mg of sodium carboxymethyl cellulose, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel A;

[0100] (2) Weigh 250.0 mg of sodium carboxymethyl cellulose, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel B;

[0101] (3) Weigh 375.0 mg of sodium carboxymethyl cellulose, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel C;

[0102] (4) Weigh 625.0 mg of hydroxypropyl methylcellulose, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel D;

[0103] (5) Weigh 750.0 mg of hydroxypropyl methylcellulose, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel E;

[0104] (6) Weigh 875.0 mg of hydroxypropyl methylcellulose, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel F;

[0105] (7) Weigh 125.0 mg of chitosan, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel G;

[0106] (8) Weigh 250.0 mg of chitosan, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel H;

[0107] (9) Weigh 375.0 mg of chitosan, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel I;

[0108] (10) Weigh 75.0 mg of carbomer 940, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel J;

[0109] (11) Weigh 100.0 mg of carbomer 940, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel K;

[0110] (12) Weigh 125.0 mg of carbomer 940, add 10 mL of distilled water and 2.5 mL of propylene glycol, stir at 1000 r / min overnight to fully swell, and obtain gel L;

[0111] B. Add a certain amount of triethanolamine to the gel AL obtained in step A and adjust the pH of the gel to between 6.0 and 7.5.

[0112] 3.2 Viscosity Measurement Results of Different Blank Gel Agents

[0113] The viscosity determination results of blank gels prepared from different gel matrices are shown in Table 7.

[0114] Table 7 Viscosities of different blank gels in Example 3

[0115]

[0116] Table 7 shows that, compared to other gel matrices, the gel prepared with Carbomer 940 exhibits higher viscosity even at low concentrations. Although the concentrations of the other three gel matrices were higher than those of the Carbomer 940 group, the maximum viscosity of the gels prepared with them was significantly lower than that prepared with 0.6 wt% Carbomer 940. Carbomer-based gels exhibit good stability within the pH range of 5.0-11.0, are non-irritating to skin and mucous membranes, do not decompose under high temperatures, and can withstand autoclaving, meeting the practical production requirements of gels.

[0117] Example 4: Screening of the ratio of gel matrix to humectant and viscosity determination

[0118] 4.1 Preparation of compound nanogels with different proportions of natural active ingredients

[0119] Using carbomer 940 as the gel matrix and propylene glycol as the humectant, the pre-screening ratio of the two is shown in Table 8, and nanogels with different ratios of natural active ingredients were prepared accordingly.

[0120] Table 8. Pre-screening ratio of carbomer 940 and propylene glycol

[0121]

[0122] The specific preparation steps are as follows:

[0123] S1. Add 330.0 mg of magnolol and 660.0 mg of glyceryl laurate to 10 mL of anhydrous ethanol and sonicate for 10 min to promote dissolution.

[0124] S2. Stir at 80℃ and 2400r / min for 90min, adding 15mL of distilled water during the process. In the last 30min, open the stopper to evaporate the ethanol, and finally obtain the natural active ingredient nano suspension A.

[0125] S3、(1) Weigh 180.0 mg of carbomer 940, add 1.5 mL of propylene glycol, add distilled water to make up the volume to 15 mL, stir at 1000 r / min overnight to fully swell, and obtain gel A;

[0126] (2) Weigh 180.0 mg of carbomer 940, add 3 mL of propylene glycol, and make up the volume to 15 mL with distilled water. Stir at 1000 r / min overnight to fully swell and obtain gel B.

[0127] (3) Weigh 180.0 mg of carbomer 940, add 4.5 mL of propylene glycol, and make up the volume to 15 mL with distilled water. Stir at 1000 r / min overnight to fully swell and obtain gel C.

[0128] (4) Weigh 240.0 mg of carbomer 940, add 1.5 mL of propylene glycol, and make up the volume to 15 mL with distilled water. Stir at 1000 r / min overnight to fully swell and obtain gel D;

[0129] (5) Weigh 240.0 mg of carbomer 940, add 3 mL of propylene glycol, and make up the volume to 15 mL with distilled water. Stir at 1000 r / min overnight to fully swell and obtain gel E.

[0130] (6) Take 240.0 mg of carbomer 940, add 4.5 mL of propylene glycol, and make up the volume to 15 mL with distilled water. Stir at 1000 r / min overnight to fully swell and obtain gel F;

[0131] (7) Weigh 300.0 mg of carbomer 940, add 1.5 mL of propylene glycol, and make up the volume to 15 mL with distilled water. Stir at 1000 r / min overnight to fully swell and obtain gel G;

[0132] (8) Weigh 300.0 mg of carbomer 940, add 3 mL of propylene glycol, and make up the volume to 15 mL with distilled water. Stir at 1000 r / min overnight to fully swell and obtain gel H.

[0133] (9) Take 300.0 mg of carbomer 940, add 4.5 mL of propylene glycol, and make up the volume to 15 mL with distilled water. Stir at 1000 r / min overnight to fully swell and obtain gel I;

[0134] S3. Add natural active ingredient nano suspension A to gel AI after it has fully swollen in S2, stir thoroughly at 1000 r / min for 5 h, and then slowly add triethanolamine to adjust the pH of the gel to between 6.0 and 7.5.

[0135] Viscosity measurement procedure:

[0136] (1) Select a 40.0mm aluminum plate as the fixture and set the fixture gap to 1000μm. Select "Peak Hold" mode, set the temperature to 25℃, the process duration to 300sec, the shear rate to 0.63rad / s, and the acquisition frequency to acquire one point every 1sec.

[0137] (2) Take a certain amount of gel and place it in the center of the clamp under the rheometer. Adjust the clamp gap to 1050 μm and start the measurement.

[0138] 4.2 Viscosity determination and results of compound nanogels with different proportions of natural active ingredients

[0139] The viscosity determination results of the natural active ingredient compound nanogels prepared with different ratios of carbomer 940 and propylene glycol are shown in Table 9.

[0140] Table 9 Viscosity of the natural active ingredient compound nanogel in Example 4

[0141]

[0142] Table 9 shows that the viscosity of the natural active ingredient compound nanogel increases with increasing carbomer 940 concentration. When the carbomer 940 concentration is 0.6 wt%, the viscosity of the natural active ingredient compound nanogel is significantly affected by the propylene glycol concentration; while when the carbomer 940 concentration is 0.8 wt% or 1.0 wt%, the viscosity is less affected by the propylene glycol concentration. Furthermore, during the gel preparation process, stirring the gel matrix is ​​difficult when the carbomer 940 concentration is 1.0 wt%, and the natural active ingredient nano-suspension is difficult to mix evenly after addition.

[0143] Therefore, 0.8wt% carbomer 940 was selected as the gel dosage for the natural active ingredient compound nanogel, and 15% propylene glycol was selected as the moisturizer dosage for the natural active ingredient compound nanogel.

[0144] Example 5: Screening of Preservative Dosage

[0145] 5.1 Preparation of compound nanogels containing different concentrations of preservatives and natural active ingredients

[0146] Using ethylparaben as a preservative, compound nanogels containing 0 wt%, 0.003 wt%, 0.03 wt%, and 0.3 wt% of preservative were prepared, and their antibacterial efficacy was tested. Table 10 shows the amount of ethylparaben added in the compound nanogels corresponding to different preservative concentrations.

[0147] Table 10. Corresponding amounts of ethylparaben in glyceryl laurate and magnolol gels with different preservative concentrations.

[0148]

[0149] Preparation steps:

[0150] (1) Add 330.0 mg of magnolol and 660.0 mg of glyceryl laurate to 10 mL of anhydrous ethanol and sonicate for 10 min to promote dissolution.

[0151] (2) Stir at 80℃ and 2400r / min for 90min, adding 15mL of distilled water during the process. In the last 30min, open the stopper to evaporate the ethanol and finally obtain the natural active ingredient nano suspension.

[0152] (3) Add ethylparaben to 0.5 mL of propylene glycol according to the dosage in Table 10 above, and dissolve by sonication to obtain solutions A, B, C and D respectively.

[0153] (4) Dissolve 0.24g of carbomer 940 in an aqueous solution, and after it swells completely, add it to the natural active ingredient nano suspension, and add solution AD separately. Stir thoroughly at 1000r / min for 5h. Then slowly add triethanolamine to adjust the pH of the gel to between 6.0 and 7.5.

[0154] 5.2 Results of antibacterial efficacy test

[0155] The antibacterial efficacy of glyceryl laurate and magnolol gels containing preservative concentrations of 0 wt%, 0.003 wt%, 0.03 wt%, and 0.3 wt% was tested, and the results are shown in Table 11.

[0156] Table 11 Antibacterial efficacy of glyceryl laurate and magnolol gel in Example 5

[0157]

[0158] Table 11 shows that when the concentration of ethylparaben is 0.03%, the inhibitory effect of glyceryl laurate / honokiol gel on Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa reaches the "A" level in the criterion for judging the antibacterial efficacy of uterine preparations. That is, compared with the same amount added, the bacterial lg (value in parentheses) value decreases by more than 2 at 6 hours and by more than 3 at 24 hours, and the fungal lg value decreases by more than 2 at 7 days.

[0159] Ultimately, 0.03% ethylparaben was chosen as the preservative dosage for the natural active ingredient compound nanogel.

[0160] Experimental Example 1: Preparation and Characterization of Nano-Suspension of Natural Active Ingredients

[0161] 1.1 Preparation of nano-suspensions of natural active ingredients

[0162] (1) Add 330.0 mg of magnolol and 660.0 mg of glyceryl laurate to 10 mL of anhydrous ethanol and sonicate for 10 min to promote dissolution.

[0163] (2) Stir at 80℃ and 2400r / min for 90min, adding 15mL of distilled water during the process. In the last 30min, open the stopper to evaporate the ethanol.

[0164] 1.2 Characterization of Natural Active Ingredient Nanosuspensions

[0165] 1.2.1 Materials and Methods

[0166] (1) Drug: Natural active ingredient nano suspension

[0167] (2) Instruments: Scanning electron microscope (SEM), transmission electron microscope (TEM).

[0168] (4) Test methods:

[0169] SEM observation: Dilute the natural active ingredient nano-suspension 10 times, drop 1 drop onto a smooth, clean aluminum foil, and let it dry. Cut the dried aluminum foil into small pieces, attach them to the stage, and perform gold spraying. After gold spraying, place the stage in the SEM for observation.

[0170] TEM observation: After diluting the natural active ingredient nano-suspension 10 times, a small amount of sample was scooped up with a copper mesh and dried. The dried copper mesh was then placed inside the TEM for observation.

[0171] 1.2.2 Results

[0172] Morphological characteristics of nanoparticles in natural active ingredient nanosuspensions, such as shape, size, and surface morphology, are as follows: Figure 2 As shown in Table 12, the PDI and particle size detection results are as follows.

[0173] Table 12. PDI and particle size of natural active ingredient nanoparticles in Experiment Example 1

[0174]

[0175] from Figure 2 As can be seen, the glyceryl laurate / honokiol nanoparticles prepared by this invention are irregularly spherical under an electron microscope, with a rough and uneven surface and a particle size between 150-250 nm.

[0176] As shown in Table 12, the average particle size of the natural active ingredient nanoparticles is 187.269±11.439 nm, and the PDI is 0.449±0.093. This result indicates that the natural active ingredient nanoparticles prepared in this invention have a relatively small and uniform particle size.

[0177] Experimental Example 2: Preparation and Characterization of Compound Nanogels Containing Natural Active Veterinary Ingredients

[0178] 2.1 Preparation of veterinary natural active ingredient nanogels

[0179] (1) Add 330.0 mg of magnolol and 660.0 mg of glyceryl laurate to 10 mL of anhydrous ethanol and sonicate for 10 min to promote dissolution.

[0180] (2) Stir at 80℃ and 2400r / min for 90min, adding 15mL of distilled water during the process. In the last 30min, open the stopper to evaporate the ethanol and obtain a nano suspension of natural active ingredients.

[0181] (3) Weigh 9.0 mg of ethylparaben, add 0.5 mL of propylene glycol, and dissolve by sonication to obtain solution A.

[0182] (4) Dissolve 0.24 g of carbomer 940 in a mixed solution of 10.5 mL of distilled water and 4 mL of propylene glycol, and stir overnight at 1000 r / min to allow for full swelling. After full swelling, add the natural active ingredient nano suspension and solution A, and stir thoroughly at 1000 r / min for 5 h to obtain gel A.

[0183] (5) Slowly add triethanolamine to gel A and stir until the pH of the gel is adjusted to between 6.0 and 7.5.

[0184] 2.2 Characterization Results

[0185] The appearance characteristics of the compound nanogel containing natural active ingredients for veterinary use are shown in the figure. Figure 3 The results of pH, viscosity, and particle size tests are shown in Table 13.

[0186] Table 13 pH, viscosity, and particle size of the veterinary natural active ingredient nanogel in Experiment Example 2

[0187]

[0188] The veterinary natural active ingredient compound nanogel prepared by this invention is a milky white, opaque, viscous semi-solid with a uniform and delicate texture, no phase separation, suitable viscosity, and good extensibility. Its pH is 6.60±0.15, viscosity is 32.123±0.907 Pa·s, and the particle size of the contained natural active ingredient nanoparticles is 193.263±30.631 nm.

[0189] Experimental Example 3: In vitro cumulative release of veterinary natural active ingredient compound nanogel

[0190] 3.1 Materials and Methods

[0191] (1) Drugs: Natural active ingredient nano suspension and veterinary natural active ingredient compound nano gel prepared in Experimental Example 2.

[0192] (2) Instruments: Dissolution apparatus, gas chromatograph.

[0193] (3) Test methods:

[0194] 4.0 g of nanogel (or 2 mL of natural active ingredient nanosuspension) was placed in a dialysis bag, sealed, and then placed in a dissolution vessel containing 250 mL of release medium (pH = 6.0). An in vitro release test was conducted at 37 ± 0.5 °C and a rotation speed of 50 r / min. 1 mL of dialysate was collected at 0.5 h, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h, 60 h, and 72 h, with 1 mL of buffer solution added simultaneously. The dialysate was then reconstituted with 1 mL of acetonitrile after nitrogen blowing, filtered through a 0.22 μm needle filter, transferred to a sample vial, and analyzed by gas chromatography. The release amounts of the nanogel and natural active ingredient nanosuspension at different time points were calculated based on the results.

[0195] 3.2 Results

[0196] The results of the in vitro cumulative release test of veterinary natural active ingredient nanogel and natural active ingredient compound nanosuspension in a simulated uterine environment (pH=6.0) are shown in Table 14 and 14. Figure 4 .

[0197] Table 14 In vitro release of different formulations in a simulated uterine environment

[0198]

[0199] Note: Blank spaces in the table indicate that the equilibrium point has been reached, and the subsequent release amount remains almost unchanged.

[0200] From Table 14 and Figure 4 It can be seen that in a simulated uterine environment (pH=6.0), the cumulative release of the natural active ingredient nano-suspension is relatively rapid, exceeding 90% release within 24 hours, indicating that nanoparticles alone do not possess significant sustained-release properties in a simulated uterine environment. However, the veterinary natural active ingredient compound nanogel prepared in this invention exhibits a sustained-release time exceeding 40 hours in a simulated uterine environment, with only 65% ​​release within 24 hours, demonstrating its superior sustained-release capability. This release characteristic will undoubtedly facilitate the absorption of more natural active ingredient nanoparticles at the uterine lesion site, thereby maximizing the therapeutic effect.

[0201] Experiment 4: Treatment of an Infection Model with Veterinary Natural Active Ingredient Nanogel

[0202] 4.1 Materials and Methods

[0203] (1) Experimental animals: 35 rats, 600g-750g, were housed in separate cages at the Experimental Animal Center of Huazhong Agricultural University.

[0204] (2) Test methods

[0205] The experiment was conducted in strict accordance with animal welfare principles, ensuring suitable temperature, sufficient feed, and free access to water. Six rats were selected as the normal control group, and the remaining rats were used as the infection model group. Each rat was injected intramuscularly with 0.15 mg / (kg·d) of estradiol benzoate for 3 consecutive days to induce estrus. After 3 days, 0.3 mL of 3% glacial acetic acid was instilled into the rat endometrium to stimulate the endometrium. In the normal control group, 0.3 mL of physiological saline was injected into the uterus through the cervix using a uterine instillation needle on the 3rd day after stimulation, once a day for 5 consecutive days. In the infection model group, 0.3 mL of mixed bacterial solution was injected into the uterus through the cervix using a uterine instillation needle on the 3rd day after stimulation. After administration, the rats were inverted for 2 minutes to ensure complete entry of the bacterial solution into the uterus, once a day for 5 consecutive days.

[0206] Rats with established endometritis models were regrouped into four groups, including a normal control group: normal control group, infection control group, raw material and drug physical mixture group, and natural active ingredient compound nanogel group. The normal control group received no treatment; the infection control group received no treatment after model establishment; the raw material and drug physical mixture group received 0.3 mL of a physically mixed raw material solution (66 mg laurate and 33 mg magnolol added to 3 mL distilled water and mixed thoroughly) every 2 days after model establishment, for a total of 4 times; the natural active ingredient compound nanogel group received 0.3 g (approximately 0.3 mL) of a veterinary natural active ingredient compound nanogel (approximately 6.6 mg laurate and 3.3 mg magnolol) every 2 days after model establishment, for a total of 4 times.

[0207] 4.2 Results

[0208] Table 15 shows the changes in body temperature of rats in each group during the treatment trial. The results of necropsy examinations of rat uterine tissue on days 4 and 8 of treatment are shown in [Table 15]. Figure 5 The bacterial load in uterine tissue is shown in Table 16, and the levels of pro-inflammatory cytokines in uterine tissue are shown in Table 17. The bacterial load in the uterine tissue of rats in each group after treatment is as follows: Figure 6 As shown, the expression levels of inflammatory factors in the uterine tissue of rats in each group after treatment are as follows: Figure 7 As shown, the pathological results of uterine tissue sections observed on the 8th day of treatment are as follows: Figure 8 .

[0209] Table 15 Changes in Body Temperature

[0210]

[0211] Table 15 shows that after the start of administration, the body temperature of rats in both the raw material drug-physical mixture group and the natural active ingredient compound nanogel group decreased, indicating that both drugs could reduce the high temperature induced by endometritis. The natural active ingredient nanogel group showed a faster rate of temperature decrease, followed by the raw material drug-physical mixture group, suggesting that the raw material drug-physical mixture was less effective than the natural active ingredient compound nanogel.

[0212] Table 16 Bacterial load in rat uterine tissue on days 4 and 8

[0213]

[0214] Table 16 shows that there were almost no bacteria in the uterus of the normal control group rats, while the bacterial count in the uterus of the infected control group rats exceeded 1.5 × 10⁻⁶. 4 CFU / mL. The bacterial content in the uterus of rats in the treatment group was significantly reduced, and the bacterial content in the uterus of rats in the natural active ingredient nanogel group was lower than that in the raw material and drug mixture group. This result indicates that the veterinary natural active ingredient nanogel has a strong ability to inhibit bacterial growth in the uterus.

[0215] Table 17 Levels of pro-inflammatory cytokines in rat uterine tissue on days 4 and 8

[0216]

[0217] Table 17 shows that on day 4 of treatment, the levels of all pro-inflammatory cytokines in the raw material / pharmaceutical mixture group were higher than those in the natural active ingredient nanogel group. On day 8 of treatment, the levels of all pro-inflammatory cytokines in the raw material / pharmaceutical mixture group remained high, while the expression levels of IL-6, IL-1β, TNF-α, and IL-8 in the natural active ingredient nanogel group were close to those in the normal control group. These results indicate that veterinary natural active ingredient nanogels can effectively inhibit the expression of pro-inflammatory cytokines in tissues.

[0218] Depend on Figure 5 It was found that, compared with the normal control group, the uterus of the infected control group rats was significantly enlarged, with fluid accumulation inside, and in severe cases, even adhesion to surrounding tissues. On the 4th day of treatment, the fluid accumulation in the uterus of the treated rats decreased, and the degree of swelling was reduced. On the 8th day of treatment, the uterus of the rats in the raw material and drug combination group had slight swelling, while the uterine swelling of the rats in the natural active ingredient nanogel group had basically disappeared, indicating that the natural active ingredient nanogel can effectively relieve the swelling caused by endometritis.

[0219] Depend on Figure 6It can be seen that there were almost no bacteria in the uterus of the normal control group rats, while the bacterial count in the uterus of the infected control group rats exceeded 1.5 × 10⁻⁶. 4 CFU / mL. The bacterial content in the uterus of rats in the treatment group was significantly reduced, and the bacterial content in the uterus of rats in the natural active ingredient nanogel group was lower than that in the raw material drug mixture group. These results indicate that the natural active ingredient nanogel has a strong ability to inhibit bacterial growth in the uterus.

[0220] Depend on Figure 7 It was found that on days 4 and 8 of treatment, the levels of all pro-inflammatory cytokines in the raw material / pharmaceutical mixture group were higher than those in the natural active ingredient nanogel group. On day 8 of treatment, the expression levels of IL-6, TNF-α, and IL-8 in the natural active ingredient nanogel group were close to those in the normal control group. These results indicate that the natural active ingredient nanogel has a strong inhibitory effect on the expression of inflammatory mediators IL-6, TNF-α, and IL-8.

[0221] Depend on Figure 8 It was found that the endometrial structure of the rats in the blank control group was intact, with no inflammatory cell infiltration. The endometrial structure of the rats in the infection control group was significantly damaged, with some single-layer columnar epithelial cells sloughing off, and the deeper cells showing a swollen and stretched state, with a large number of neutrophils observed. The number of inflammatory granulocytes was reduced and the endometrial structure was improved in the raw material / pharmaceutical mixture group and the natural active ingredient nanogel group.

[0222] In summary, this invention successfully prepared a veterinary natural active ingredient nanogel with highly efficient antibacterial and anti-inflammatory capabilities. Its strong sustained-release ability is conducive to maximizing the efficacy of the natural active ingredient and also beneficial for its application in veterinary clinical practice for the prevention and control of endometritis.

[0223] This invention discloses a natural active ingredient compound nanogel composed of natural active ingredients, a gel matrix, and excipients. The formulation is rational, the process is simple, the properties are stable, it is convenient to use, and it is safe and long-lasting. This natural active ingredient compound nanogel contains two synergistic natural active ingredients that can inhibit the proliferation of pathogenic bacteria causing endometritis, such as Staphylococcus aureus and Streptococcus, and reduce the expression of pro-inflammatory cytokines at the site of inflammation, showing great promise for clinical use. Simultaneously, it has a certain sustained-release capability; the slowly released natural active ingredient nanoparticles, due to their small particle size, are more easily taken up by cells, enabling prolonged drug residence and absorption at the administration site, thereby enhancing the duration of action, reducing the frequency of administration, and improving efficacy.

[0224] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. The application of a compound nanogel containing natural active ingredients for veterinary use in the preparation of drugs for treating uterine bacterial infections or in the preparation of drugs for alleviating uterine bacterial inflammatory infections, characterized in that, The veterinary natural active ingredient compound nanogel is made of natural active ingredients, gel matrix and additives; The natural active ingredient is composed of a first natural active ingredient and a second natural active ingredient; the first natural active ingredient is glyceryl laurate; the second natural active ingredient is honokiol; the mass ratio of the first natural active ingredient to the second natural active ingredient is (1-2):1; the amount of the natural active ingredient is 3-4 wt%. The gel matrix is ​​Carbomer 940, used in an amount of 0.8 wt%. The additives are propylene glycol and ethylparaben, wherein the amount of propylene glycol is 15 wt% and the amount of ethylparaben is 0.03 wt%. The preparation method of the veterinary natural active ingredient compound nanogel includes the following steps: adding the natural active ingredient to anhydrous ethanol, ultrasonically treating it, and preparing a natural active ingredient nano suspension by hydrothermal method; then, after the gel matrix is ​​fully swollen, adding it and the additives to the nano suspension, stirring, and adjusting the pH to 6.0-7.5 to obtain the veterinary natural active ingredient compound nanogel; the hydrothermal method is carried out at 80℃ and 2400r / min, with distilled water added successively during the process, and the bottle stopper is opened at the end to evaporate the ethanol.

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